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Aim Inactivated Orf virus ( ORFV, Parapoxvirus ovis) demonstrates strong antiviral activity in animal models including a human hepatitis B virus ( HBV)-transgenic mouse. In addition, expression of interferon ( IFN)-γ and interleukin-10 ( IL-10) was induced after administration of inactivated ORFV in these mice. IFN-γ and IL-10 are known to elicit antifibrotic activity. We therefore aimed to study antifibrotic activity of inactivated ORFV in models of liver fibrosis. Methods We characterized ORFV-induced hepatic cytokine expression in rats. We then studied ORFV in two models of liver fibrosis in rats, pig serum-induced liver fibrosis and carbon tetrachloride ( CCL4)-induced liver fibrosis. Results ORFV induced hepatic expression of IFN-γ and IL-10 in rats. ORFV mediated antifibrotic activity when administrated concomitantly with the fibrosis-inducing agents in both models of liver fibrosis. Importantly, when CCL4-induced liver fibrosis was already established, ORFV application still showed significant antifibrotic activity. In addition, we were able to demonstrate a direct antifibrotic effect of ORFV on stellate cells. Conclusion These results establish a potential novel antifibrotic therapeutic approach that not only prevents but also resolves established liver fibrosis. Further studies are required to unravel the details of the mechanisms involved. [ABSTRACT FROM AUTHOR]

Development of tumor-specific T cell tolerance contributes to the failure of the immune system to eliminate tumor cells. Here we report that hematogenous dissemination of tumor cells followed by their elimination and local removal of apoptotic tumor cells in the liver leads to subsequent development of T cell tolerance towards antigens associated with apoptotic tumor cells. We provide evidence that liver sinusoidal endothelial cells (LSEC) remove apoptotic cell fragments generated by induction of tumor cell apoptosis through hepatic NK1.1 cells. Antigen associated with apoptotic cell material is processed and cross-presented by LSEC to CD8 T cells, leading to induction of CD8 T cell tolerance. Adoptive transfer of LSEC isolated from mice challenged previously with tumor cells promotes development of CD8 T cell tolerance towards tumor-associated antigen in vivo. Our results indicate that hematogenous dissemination of tumor cells, followed by hepatic tumor cell elimination and local cross-presentation of apoptotic tumor cells by LSEC and subsequent CD8 T cell tolerance induction, represents a novel mechanism operative in tumor immune escape. [ABSTRACT FROM AUTHOR]

Background Recombinant replication-deficient adenoviral vectors (recAd) are attractive candidates for DNA vaccination approaches because they are able to activate the innate and adaptive immune systems. Here we explore the ability of recAd to transduce and activate subsets of dendritic cells, namely plasmacytoid dendritic cells (pDC) and conventional dendritic cells (cDC). Methods DC were derived from bone marrow precursors in vitro with the help of FLT3-ligand. Sorted populations of pDC and cDC were infected with recAd at various multiplicities of infection. Transduction efficiency, phenotypic maturation and production of IFN-α as well as IL-6 were assessed. Additionally, activation of DC and induction of cytotoxic T lymphocytes (CTL) were determined in vivo. The role of Toll-like receptor (TLR) 9 in recAd recognition was investigated as it has previously been shown that DNA viruses are recognized via this receptor. Results RecAd can efficiently transduce pDC as well as cDC in vitro. Both DC subsets mature and produce IFN-α upon interaction with recAd. In the absence of TLR9, activation and cytokine production was only detected in cDC but not in pDC. Importantly, induction of CD8+ CTL following in vivo injection of recAd was similar in TRL9-deficient mice when compared with wildtype controls. Conclusions RecAd can efficiently transduce and activate both pDC and cDC. pDC required TLR9 to detect the presence of recAd whereas cDC also recognized recAd independently of TLR9. These unique immunostimulatory properties support the future development of recombinant Ad as a vector for DNA vaccine approaches. Copyright © 2006 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Recombinant DNA vaccines are able to induce strong CD8+ T cell mediated immunity and have become increasingly attractive for the prevention and treatment of infectious diseases and cancer. Dendritic cells (DC), which critically control cellular immune responses, have been transduced with antigen ex vivo and used as 'nature's adjuvant' to enhance vaccine efficacy. The impact of the application route on the in vivo distribution of antigen and the stimulation of CD8+ T cells have been subjects of considerable debate. Here we report the construction of vectors expressing a fusion protein between EGFP, the H2-Kb-binding peptide OVAaa257−264 and green click beetle luciferase as a model antigen which allows for simultaneous quantitative assessment of antigen expression using fluorescence and bioluminescence imaging in correlation with CD8+ T cell stimulation in vivo. We applied this construct to evaluate DNA vaccination with recombinant adenoviral vectors, assess the impact of using cultured DC for vaccine delivery and investigate different application routes. Antigen expression was non-invasively followed in vivo by visualizing bioluminescence with an ultrasensitive CCD camera. CD8+ T cell stimulation was detected with H2-Kb-OVAaa257−264 tetramers. We found that intravenous injection of adenovirus-transduced DC stimulated the strongest OVAaa257−264-specific cytotoxic T-lymphocyte (CTL) responses although it delivered two orders of magnitude less antigen in vivo when compared to direct injection of recombinant adenovirus. We believe that our experimental approach has the potential to facilitate translational development of improved genetic immunization strategies targeting DC directly in vivo. Copyright © 2006 John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Only recently have natural antigens for CD1d-dependent, invariant Vα14 natural killer T ( iNKT) cells been identified. Similar data for CD1d-independent and CD8 NKT cell populations are still missing. Here, we show that the MHC class I-restricted CD8 TCR-transgenic mouse lines OT-I, P14 and H-Y contain a significant proportion of transgenic CD8 NK1.1 T cells. In liver, most of NK1.1 T cells express CD8αα homodimers. Transgenic NKT cells did not bind invariant Vα14-to-Jα18 TCR rearrangement (Vα14 i)-specific CD1d/α-galactosylceramide tetramers and the frequency of iNKT cells was severely reduced. The activated cell surface phenotype and the distribution of transgenic NKT cells in vivo were similar to that reported for iNKT cells. The OT-I and P14 CD8 NKT cells recognized their cognate antigen in the context of H2-K and produced cytokines shortly after TCR stimulation. Importantly, transgenic NKT cells exerted immediate antigen-specific cytotoxicity in vitro and in vivo. Our results demonstrate the presence of transgenic CD8 NKT cells in MHC class I-restricted TCR-transgenic animals, which are endowed with rapid antigen-specific effector functions. These data imply that experiments studying naive T cell function in TCR-transgenic animals should be interpreted with caution, and that such animals could be utilized for studying CD8 NKT cell function in an antigen-specific manner. [ABSTRACT FROM AUTHOR]

Dissemination of soluble molecules or antigens via the blood stream is considered to lead to a uniform distribution in the various organs of the body , but organ-specific microarchitecture and vascularization may influence this. Following intravenous injection of αCD3ε antibody (αCD3εAb) we observed clear differences in antibody binding to Fcγ receptor (FcγR)+ antigen-presenting cells (APCs) or T lymphocytes in different organs. Significant binding of blood-borne αCD3εAb was only detected in the spleen and liver and not in the thymus or lymph node. In the spleen, only 10% of dendritic cells/macrophages and 40% of T-cell receptor (TCR)-β+ cells were positive for αCD3εAb, and, dependent on FcγR-mediated cross-linking of αCD3εAb, a similar percentage of splenic TCR-β+ cells were stimulated and became CD69+. Stimulation of TCR-β+ cells in the liver was at least as efficient as in the spleen, but almost all T cells and all scavenger liver sinusoidal endothelial cells bound αCD3εAb. In contrast to CD69 up-regulation, only CD4+ natural killer T (NKT) cells and CD11ahigh CD8+ T cells were activated by αCD3εAb and expressed interferon (IFN)-γ. Again, IFN-γ release from NKT/T cells was at least as efficient in the liver as in the spleen. Taken together, our results support the notion that the combination of extensive hepatic vascularization and very high scavenger activity allows the liver to fulfill its metabolic tasks and to promote stimulation of the large but widely distributed hepatic population of NKT/T cells. [ABSTRACT FROM AUTHOR]

CpG-rich oligonucleotides (CpG-ODN) bind to Toll-like receptor 9 (TLR9) and are used as powerful adjuvants for vaccination. Here we report that CpG-ODN not only act as immune stimulatory agents but can also induce strong immune suppression depending on the anatomical location of application. In agreement with the adjuvant effect, subcutaneous application of antigen plus CpG-ODN resulted in antigen-specific T cell activation in local lymph nodes. In contrast, systemic application of CpG-ODN resulted in suppression of T cell expansion and CTL activity in the spleen. The suppressive effect was mediated by indoleamine 2,3-dioxygenase (IDO) as indicated by the observation that CpG-ODN induced IDO in the spleen and that T cell suppression could be abrogated by 1-methyl-tryptophan (1-MT), an inhibitor of IDO. No expression of IDO was observed in lymph nodes after injection of CpG-ODN, explaining why suppression was restricted to the spleen. Studies with a set of knockout mice demonstrated that the CpG-ODN-induced immune suppression is dependent on TLR9 stimulation and independent of type I and type II interferons. The present study shows that for the use of CpG-ODN as an adjuvant in vaccines, the route of application is crucial and needs to be considered. In addition, the results indicate that down-modulation of immune responses by CpG-ODN may be possible in certain pathological conditions. See accompanying commentary: [ABSTRACT FROM AUTHOR]

After ingestion, oral antigens distribute systemically and provoke T cell stimulation outside the gastrointestinal tract. Within the liver, scavenger liver sinusoidal endothelial cells (LSEC) eliminate blood-borne antigens and induce T cell tolerance. Here we investigated whether LSEC contribute to oral tolerance. Oral antigens were efficiently cross-presented on H-2K by LSEC to naive CD8 T cells. Cross-presentation efficiency in LSEC but not dendritic cells was increased by antigen-exposure to heat or low pH. Mechanistically, cross-presentation in LSEC requires endosomal maturation, involves hsc73 and proteasomal degradation. H-2K-restricted cross-presentation of oral antigens by LSEC in vivo induced CD8 T cell priming and led to development of CD8 T cell tolerance in two independent experimental systems. Adoptive transfer of LSEC from mice fed with antigen (ovalbumin) into RAG2 knockout mice, previously reconstituted with naive ovalbumin-specific CD8 T cells, prevented development of specific cytotoxicity and expression of IFN-γ in CD8 T cells. Using a new transgenic mouse line expressing H-2K only on endothelial cells, we have demonstrated that oral antigen administration leads to tolerance in H-2K-restricted CD8 T cells. Collectively, our data demonstrate a participation of the liver, in particular scavenger LSEC, in development of CD8 T cell tolerance towards oral antigens. [ABSTRACT FROM AUTHOR]

Establishment of self-tolerance prevents autoaggression against organ-specific self-antigents. This beneficial effect, however, may in turn be responsible for tumor immune evasion. Thus, dissecting the mechanisms leading to the breakdown of self-tolerance in autoimmune diseases might provide insights for successful antitumor immune therapies. In a variety of animal models, organ- or tumor-specific immunity has been described, focusing on antigen-specific T-cell activation. Here, we discuss two transgenic mouse models which demonstrate that both autoaggression and tumor rejection require more than activated, self-reactive T cells. TCR transgenic mice, which are tolerant to a liver-specific MHC class I antigen, K[supb], can be activated to reject K[supb]-positive grafts, but fail to attack K[supb]-expressing liver. However, autoaggression occurs when activated T cells are combined with "conditioning" of the target organ by irradiation or infection with a liver-specific pathogen. Similarly, in a mouse model of islet cell carcinoma, neither co-stimulatory tumor cells nor highly activated antitumor lymphocytes provoke an effective immune response against the tumor. Instead, a combination of activated lymphocytes and irradiation is required for lymphocyte infiltration into solid tumors. Both model systems provide evidence that although activated antigen-specific lymphocytes are a prerequisite for autoaggression, effector cell extravasation and appropriate interaction with the target organ/tumor are equally important. Thus, we propose that the organ/tumor microenvironment is a critical parameter in determining the effectiveness of an anti-self immune response. [ABSTRACT FROM AUTHOR]